Profiling of gene expression in the brain associated with anxiety-related behaviors in the chronic phase following cranial irradiation

Although the brain is exposed to cranial irradiation in many clinical contexts, including malignant brain tumor therapy, such exposure can cause delayed neuropsychiatric disorders in the chronic phase. However, how specific molecular mechanisms are associated with irradiation-induced behavioral dysfunction, especially anxiety-like behaviors, is unclear. In the present study, we evaluated anxiety-like behaviors in adult C57BL/6 mice using the open-field (OF) and elevated plus maze (EPM) tests 3 months following single cranial irradiation (10 Gy). Additionally, by using RNA sequencing (RNA-seq), we analyzed gene expression profiles in the cortex and hippocampus of the adult brain to demonstrate the molecular mechanisms of radiation-induced brain dysfunction. In the OF and EPM tests, mice treated with radiation exhibited increased anxiety-like behaviors in the chronic phase. Gene expression analysis by RNA-seq revealed 89 and 106 differentially expressed genes in the cortex and hippocampus, respectively, following cranial irradiation. Subsequently, ClueGO and STRING analyses clustered these genes in pathways related to protein kinase activity, circadian behavior, and cell differentiation. Based on our expression analysis, we suggest that behavioral dysfunction following cranial irradiation is associated with altered expression of Cdkn1a, Ciart, Fos, Hspa5, Hspb1 and Klf10. These novel findings may provide potential genetic targets to investigate for the development of radioprotective agents.

www.nature.com/scientificreports/ To demonstrate how cranial irradiation affects brain functions, we investigated anxiety-like behavior in C57BL/6 mice at 3 months after cranial irradiation. In addition, we investigated the gene expression profiles in the cortex and hippocampus of adult mice after cranial irradiation to determine the molecular mechanisms by which ionizing radiation affects brain function and to identify novel candidate genes associated with behavioral dysfunction.

Results
Anxiety-like behavior in C57BL/6 mice in the chronic phase following cranial irradiation. A schematic diagram of this study is shown in Fig. 1A. To investigate whether cranial irradiation alters anxietyrelated behaviors in the chronic phase, we first observed the behavior of mice in an OF apparatus. Irradiated mice spent significantly less time in the center, traveled less distance, and made fewer entries into the center of the apparatus, which are indications of enhanced anxiety-like behavior (Fig. 1B,C). However, no difference in total distance traveled was evident between controls and irradiated mice at 3 months post-irradiation. A similar pattern of behavior was observed in the EPM test. Mice irradiated with 10 Gy spent significantly less time and traveled less distance in the open arms of the EPM (Fig. 1D,E). In addition, irradiated mice traveled a significantly lower distance in the EPM, indicating a reduction in locomotor activity under anxiogenic conditions (Fig. 1E). These findings indicate that cranial irradiation enhanced anxiety-like behavior in the chronic phase following cranial irradiation.

Differentially expressed genes (DEGs) in the cortex and hippocampus after cranial irradiation.
To determine the radiation-specific gene profiles related to anxiety-like behavior, total RNA-seq was carried out in the cortex and hippocampus at 3 months after cranial irradiation. As a criterion for the RNA-seq data, we assumed a fold change of 1.5 and a p-value of 0.05. Compared to their expression in the control group, 89 and 106 genes were differentially expressed in the cortex and hippocampus, respectively, of the irradiated mice ( Fig. 2A). In detail, 75 and 92 genes were uniquely dysregulated in the cortex and hippocampus, respectively, while 97 genes were upregulated and 70 genes were downregulated. Moreover, 14 genes were dysregulated in both regions, while 9 genes were upregulated and 5 genes were downregulated.
Functional gene annotation and enrichment analyses. The clustering heatmap of the 181 DEGs in the cortex and/or hippocampus at 3 months following cranial irradiation is shown in Supplementary Fig. 1. The 181 DEGs modified by cranial irradiation in the functional network were clustered by ClueGO into functional groups related to Gene Ontology (GO) biological processes, resulting in 13 terms ( Table 1). The highest number of identified genes were classified directly under negative regulation of protein kinase activity (GO:0006469, GO:0071901), followed by regulation of circadian rhythm (GO:0042752) and muscle cell migration (GO:0014812). The genes whose expression was induced by radiation were mainly associated with protein kinase activity (18.75%), circadian behavior (18.75%), and myeloid leukocyte differentiation (15.62%) (Fig. 2B,C). Other biological processes, such as corticosteroid hormone secretion, chaperone-mediated protein folding, and the response to glucocorticoids, were also represented (Fig. 2C).
To further demonstrate the perturbation induced by cranial irradiation, we examined targets of the DEGs using STRING analysis, selecting the top 10 GO terms (Fig. 3A,B). Among these, the most interesting identified GO terms were "circadian behavior", "cell differentiation", and "protein kinase activity". In the network identified with the STRING analysis, whereas 8 and 6 genes were associated with circadian rhythm (GO:0007623) and skeletal muscle cell differentiation (GO:0035914), another 7 genes displayed association with protein kinase activity (GO:0071901).
qRT-PCR validation. Since many DEGs were associated with several biological processes of the brain, the obtained RNA-seq data were validated by qRT-PCR. Notably, 30 genes clustered by both ClueGO (47 genes) and STRING (35 genes) analyses at 3 months post-irradiation were selected (Table 2). To validate the RNA-seq data demonstrated the genes that were significantly up-or downregulated by cranial irradiation in both the cortex and hippocampal tissue, we examined the gene expression levels by qRT-PCR analysis. There were 9 DEGs whose levels were significantly upregulated by radiation exposure in both the cortex and hippocampus of the mouse brain. In addition, 4 genes were significantly downregulated in the cortex and hippocampal tissue. qRT-PCR analysis confirmed that 8 and 3 genes were significantly upregulated (Fig. 4A) and downregulated (Fig. 4B), respectively, in the chronic phase following cranial irradiation. Among them, the expression patterns of 6 genes, Cdkn1a, Ciart, Fos, Hspa5, Hspb1 and Klf10, were identical in both the cortex and hippocampus, suggesting specific and chronic changes in the expression of these genes after radiation exposure. In contrast, other genes whose levels were identified as changed after cranial irradiation did not follow the same expression pattern.

Discussion
People are exposed to cranial irradiation for the treatment of many brain tumors, including primary and metastatic brain tumors and head and neck malignancies 15 . However, patients with radiotherapy are concerned about the impact of ionizing radiation because it can induce delayed cognitive and emotional dysfunction, impairing the quality of life of patients treated with radiotherapy 16 . Previous studies have provided scientific evidence of radiation-induced brain dysfunction. For example, a preclinical study demonstrated that a single dose of 10 Gy induced hippocampal-dependent behavioral dysfunctions by affecting hippocampal neurogenesis and neural plasticity-related signals 7,10 . In addition, mice showed impaired hippocampal-dependent cognitive functions at 1 month after cranial irradiation by the novel object recognition memory test; this impairment persisted up to 3 months following cranial irradiation with 10 Gy 8 . However, studies investigating the delayed effect of irradiation  Previous studies have demonstrated that cranial irradiation induced the loss of neuronal stem cells (NSCs), which underwent long-lasting changes, including apoptosis, decreased proliferation, and altered differentiation 17,18 . Rodents showed deficits in hippocampal neurogenesis, and transplanted NSCs failed to differentiate into neurons in the irradiated brain 19 . Although numerous studies have reported the effects of cranial irradiation on a variety of cellular functions, only a few studies have focused on the delayed dysregulation of gene expression in the mouse brain. The genes Csf1r, Egr1, Fos, Nr4a1, Nr4a3, and Klf10, which were found in our study to be differentially expressed after cranial irradiation, are involved in cell differentiation. Csf1r, which regulates the survival, proliferation, and differentiation of microglia 20 , was downregulated following cranial irradiation, in line with the findings that Iba1, a microglial marker, was decreased in the chronic phase 10 . Moreover, a previous study reported that Klf10 plays an important role as a tumor suppressor, and overexpression of Klf10 downregulates cell proliferation in many cancers 21 . This study showed that Klf10 expression was increased in the cortex and hippocampus following cranial irradiation, which might be associated with dysregulated cell differentiation 19 . Additionally, the radiation-induced disturbance of immediate-early genes (IEGs) in the hippocampus by contextual fear conditioning has been reported 6 . Our results showed that cranial irradiation induced disturbance in the expression of IEGs, including Egr1 and Fos, which have been implicated in neuronal plasticity and memory formation in the brain 22 . Nr4a1 and Nr4a3, nuclear receptors that function as transcription factors, have been implicated in the regulation of IEGs 23 . This is consistent with our results showing that the expression of Nr4a1 as well as Egr1 and Fos was induced following cranial irradiation.
The cellular response to radiation is complicated and involves DNA damage and the apoptosis of neuronal progenitor cells, which is partially p53-mediated 24 . Cdkn1a, an important effector of p53-mediated G1 arrest in response to many stresses, was found to be upregulated in the irradiated brain and regulated an increase in glioblastoma recurrence 25 . In addition, previous results have shown the upregulation of Gadd45a and demonstrated its potential as a biomarker for biological dosimetry in radiation therapy and early-response accidents 26 . The overexpression of Hspb1, which is involved in the metastasis and susceptibility of tumor cells, has been detected in patients with tumors 27 . A previous in vivo study demonstrated that knockdown of Hspb1 enhanced the cytotoxic  28 , indicating the involvement of Hspb1 in the resistance of tumor cells to radiotherapy. In the present study, we found that while the mRNA expression levels of Gadd45a were significantly increased in the cortex, the Cdkn1a and Hspb1 genes were upregulated in both the cortex and hippocampus in the chronic  www.nature.com/scientificreports/ phase following cranial irradiation. These data demonstrated that Cdkn1a and Hspb1 can be used as potential biomarkers for radiation-induced behavioral dysfunction, especially anxiety-like behavior, in the chronic phase. Previous studies have suggested that irradiated mice display depression-like behavior 10,29 . In addition, the disturbance of circadian rhythms was reported in patients with mood disorders 30 and anxiety disorders 31 , indicating the involvement of circadian rhythm in the pathogenesis of anxiety and mood disorders. Disruption of circadian rhythms by the injection of viral vectors induced helplessness and anxiety-like behavior in mice 32 . A previous study reported decreased levels of clock genes, including Ciart and Per2, in the brains of ketamine-treated mice 33 . In addition, the patterned expression of circadian genes, including Per2 and Nr1d1, was dysregulated in the brains of patients with major depressive disorder 34 . In the present study, downregulation of clock genes, including Ciart and Nr1d1, was observed in the brains of irradiated mice, which suggested that the disruption of genes related to circadian rhythm might be related to radiation-induced behavioral dysfunction. The physiological and behavioral significance of circadian timing is complex, and further studies are required to demonstrate time-and dose-dependent changes in genes that regulate circadian rhythms by radiation exposure.
In conclusion, we provide the first evidence that cranial irradiation induces anxiety-like behavior in mice during the chronic phase, possibly via alterations in the expression of genes in the mouse cortex and hippocampus. These molecular targets, revealed by RNA-seq, might serve as biomarkers for radiation-induced behavioral dysfunctions, especially anxiety-like disorders. Cranial irradiation affects a wide range of biological processes linked to cell differentiation, circadian behavior, and kinase activity. Consequently, we suggest that anxiety-like behavior in the chronic phase may be related to alterations in the Cdkn1a, Ciart, Fos, Hspa5, Hapb1 and Klf10 genes, and additional analysis, including WGCNA, are required to compare RNA-seq data with behavioral dysfunctions (e.g. memory impairment, depression, and anxiety) in a variety of brain regions in irradiated mice. were acclimatized for 1 week before the experiments were performed. All mice were housed in an SPF animal facility and had ad libitum access to tap water and commercial rodent chow. After acclimatization, the mice were randomly divided into the sham irradiation (0 Gy, n = 10) and irradiation (10 Gy, n = 10) groups. The Institutional Animal Care and Use Committee of KIRAMS approved the study protocol (KIRAMS-2021-0064), and experiments were conducted in accordance with the inter-nationally accepted principles for laboratory animal use and care dictated by the ARRIVE guidelines 35 . Every effort was made to minimize the number of animals used and their suffering.
Irradiation. Animals received a single dose of 10 Gy irradiation using the X-RAD 320 platform (Precision X-ray, North Branford, CT) with a dose rate of 2.0 Gy/min. Mice were irradiated to the whole brain with a 20 mm × 100 mm field size. Sham-irradiated mice were placed on an identical platform for the same duration as the irradiation group but were not irradiated.
Behavioral testing. Open  and downregulated (B) genes from RNA-seq data collected from both the cortex and hippocampus. Data are expressed as the mean ± SE (n = 5 per group). *p < 0.05, **p < 0.01, and ***p < 0.001 vs. the Con group. RNA sequencing (RNA-seq). Library preparation and sequencing. Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (New England Biolabs, Inc., UK). mRNA was isolated using the Poly(A) RNA Selection Kit (Lexogen, Inc., Austria). The isolated mRNA was used for cDNA synthesis and shearing following the manufacturer's instructions. Indexing was performed using Illumina indexes 1-12. The enrichment step was carried out using PCR. Subsequently, the libraries were checked using TapeStation HS D1000 screen tape (Agilent Technologies, Amstelveen, The Netherlands) to evaluate the mean fragment size. Quantification was performed using a library quantification kit and the StepOne Real-Time PCR System (Life Technologies, Inc., USA). High-throughput sequencing was performed as paired-end 100 sequencing using NovaSeq 6000 (Illumina, Inc., USA).
Analysis of RNA-seq data. Quality control of raw sequencing data was performed using FastQC (Simon, 2010). Reads containing adapter and low-quality reads (< Q20) were removed using FASTX_Trimmer 36 and BBMap 37 . Then, the trimmed reads were mapped to the reference genome using TopHat 38 . The Read Count (RC) data were processed based on the FPKM + geometric normalization method using EdgeR within R 39 . Fragments per kb per million reads (FPKM) values were estimated using Cufflinks 40 . Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc., Korea).
Quantitative real-time RT-PCR (qRT-PCR). cDNA was prepared using random primers (Toyobo Inc., Tokyo, Japan) according to the manufacturer's instructions and stored at -20 °C. qRT-PCR amplification was performed using PowerUP 2X SYBR Green Master Mix (Thermo Fisher Scientific) on a StepOne Real-Time PCR System (Applied Biosystems, CA, USA) according to the manufacturer's instructions. The primer sequences are shown in Table 2. All data were normalized by reference to the amplification levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene; a reference dye was included in the SYBR Master Mix. Thresholds calculated by the software were used to calculate specific mRNA expression levels using the cycle-at-threshold (Ct) method, and all results are expressed as the fold change (compared to control) in each transcript determined employing the 2 −ΔΔ CT approach.
Statistical analysis. Data are expressed as the mean ± SE. Differences between the results from the shamirradiated and 10 Gy-irradiated groups were evaluated by two-tailed Student's t tests using GraphPad Prism 9 software (GraphPad Software; San Diego, CA, USA). A p value less than 0.05 was considered to indicate statistical significance.

Data availability
RNA-seq data used in this study are deposited in the Gene Expression Omnibus (GEO, https:// www. ncbi. nlm. nih. gov/ geo/) under the accession number of GSE204993.